Pivex2.4c

E. coli DH5α was used for plasmid construction and E. coli BL21 (DE3) (Novangen, USA) as the host for making cell-free extract. pIVEX2.4c (Roche, Grenzacherstrasse, Switzerland) was used to construct cell-free expression vectors. pIVEX2.4c-AQP SS9, pETDuet-CK-T7, pUC-MjtRNA, p15a-MjpPpaRS, and pIVEX2.4c-MjpPpaRS were constructed in our previous studies.

The cDNA encoding for SnRK2.1 was provided by Professor Chen from College of Life Sciences, Northwest A&F University, China. The cDNA was amplified by PCR using LA Taq DNA polymerase (Takara, Dalian, China) and primer pairs as follows: 5′-CCGCCATGGACAAGTATGACGTTG-3′ and 5′-CGGGATCCTTAAGCTTTGTCAGACTCT-3′. Restriction sites (NcoI and BamHI) are underlined and the stop codon TAA introduced is italicized. The PCR product was digested with NcoI and BamHI restriction enzymes (Takara, Dalian, China) and ligated into the identically digested cell-free expression vector pIVEX2.4c (Roche Applied Science, Mannheim, Germany), carrying a His6-tag sequence at the N-terminal, by T4 DNA ligase (Takara, Dalian, China). The construction of the expression vector pIVEX2.4c-SnRK2.1, which encoded a His6-tag at the N-terminal of target protein, was confirmed by restriction enzyme digestion and the correctness of sequence was confirmed by DNA sequencing.